Pneumococcal vaccine containing pneumococcal surface protein A

ABSTRACT

A pneumococcal vaccine comprising a fusion protein at least comprising a full-length family 1 pneumococcal surface protein A (PspA) or a fragment thereof, and a full-length family 2 PspA or a fragment thereof, in particular any one of the following fusion proteins (1) to (3):
     (1) a fusion protein at least comprising a family 1, clade 2 PspA and a family 2, clade 3 PspA,   (2) a fusion protein at least comprising a family 1, clade 2 PspA and a family 2, clade 4 PspA, and   (3) a fusion protein at least comprising a family 1, clade 2 PspA and a family 2, clade 5 PspA,
 
is useful as a pneumococcal vaccine comprising a single protein antigen that has broadly cross-reactive immunogenicity and can induce immune response against a wide range of pneumococcal clinical isolates.

RELATED APPLICATIONS

This application is the U.S. national stage pursuant to 35 U.S.C. § 371,of U.S. international application Ser. No. PCT/JP2013/058401, filed Mar.22, 2013, designating the United States and published in English on Mar.27, 2014 as publication WO 2014/045621 A1, which claims the benefit ofJapanese application Ser. No. 2012-206039, filed Sep. 19, 2012. Theentire contents of the aforementioned patent applications areincorporated herein by this reference.

TECHNICAL FIELD

The present invention relates to a pneumococcal surface proteinA-containing pneumococcal vaccine.

BACKGROUND ART

Pneumococcus is a major respiratory tract pathogen and causes infectionsin children and adults, such as invasive pneumococcal disease (IPD)including meningitis and sepsis, and community-acquired pneumonia. Theantigens of the current pneumococcal vaccines are capsularpolysaccharides, which determine the serotypes of pneumococci, and sofar as known, there are at least 93 serotypes.

A heptavalent pneumococcal conjugate vaccine (PCV7), which is composedof a non-toxic diphtheria toxin (CRM₁₉₇) bound to polysaccharideantigens, was introduced as a pediatric vaccine in the U.S.A. in 2000.After the introduction of PCV7, the incidence of IPD caused by the sevenserotypes covered by this vaccine was clearly reduced, but an increasein the incidence of pediatric and adult IPD caused by nonvaccineserotypes such as 19A became a problem. For this reason, a 13-valentpneumococcal conjugate vaccine (PCV13), which is composed of PCV7 andadditional capsular polysaccharide antigens of six other serotypes, wasintroduced in 2010 and already approved for children and adults in theU.S.A.

However, according to a large-scale survey by Non Patent Literature 1,only 60% of pediatric invasive pneumococcal isolates collected inAlabama, U.S.A. between 2002 and 2010 (before the introduction of PCV13)had serotypes covered by PCV13. The remaining 40% of these isolatesincluded 17 serotypes that were not covered by PCV13 (Non PatentLiterature 1). In Japan, publicly-aided pediatric PCV7 vaccination wasstarted in 2011, but it was reported in 2012 that the incidence of IPDcaused by nonvaccine serotypes was increased as is the case in theU.S.A. (Non Patent Literature 2). Thus, it is unreal to continuecomplementing the current vaccines with capsular polysaccharide antigensof nonvaccine serotypes, and this implies the limitations of the currentpneumococcal vaccines based on capsular polysaccharides.

Recently, pneumococcal surface protein A (hereinafter referred to as“PspA”), which is a pneumococcal surface protein antigen, has drawnattention as a novel pneumococcal vaccine antigen to compensate for theabove-described drawback of the current pneumococcal vaccines. PspA hasa structure composed of several domains shown in FIG. 1, and theα-helical region and the proline-rich region of PspA are known to haveantigen epitopes for recognition by protective antibodies againstpneumococcal infection (Non Patent Literature 3 and 4). According to thegene sequences of the antigen epitope regions, PspA is roughly groupedinto three families including six subgroups called clades. Regarding thePspA family distribution, families 1 and 2 account for 98% or more ofpneumococcal clinical isolates (Non Patent Literature 5). PspA is knownto serve as a virulence factor to inhibit the deposition of complementC3 onto pneumococcal cells (Non Patent Literature 6), and in contrast,an anti-PspA specific antibody is known to exert protective effectagainst pneumococcal infection by antagonizing the inhibitory action ofPspA against complement deposition (Non Patent Literature 7 and 8). Thisinfection protective effect is reportedly exerted also by antibodiesthat cross-recognize different families of PspAs (Non Patent Literature9). Due to the diversity of the cross-reactive immunogenicity amongdifferent PspAs (Non Patent Literature 10 and 11), appropriate selectionof a combination of clades belonging to families 1 and 2 for broadercross-reactive immunogenicity is important in the development ofPspA-based vaccines.

The usefulness of PspA proteins as an immunogenic component of vaccinesagainst pneumococcal infection is described, for example, in PatentLiterature 1 and 2.

Non Patent Literature 12 describes the examination on the vaccineeffects of a fusion protein of a family 1, clade 1 PspA and a family 2,clade 4 PspA and a fusion protein of a family 1, clade 1 PspA and afamily 2, clade 3 PspA. Non Patent Literature 13 describes theexamination on the vaccine effect of a fusion protein of a family 1,clade 2 PspA and a family 2, clade 4 PspA. However, these PspA-basedfusion proteins described in the two cited references have not beenevaluated for the vaccine effects on pneumococcal stains expressingPspAs of clades 5 and 6, or for the vaccine effects against a wide rangeof pneumococcal clinical isolates, and also there has been no reportthat these fusion proteins are already in practical use.

CITATION LIST Patent Literature

-   Patent Literature 1: JP 6-504446 B2-   Patent Literature 2: JP 2000-503676 W

Non Patent Literature

-   Non Patent Literature 1:-   Croney C M, Coats M T, Nahm M H et al. 2012. PspA family    distribution, unlike capsular serotype, remains unaltered following    introduction of the heptavalent pneumococcal conjugate vaccine. Clin    Vaccine Immunol. 19: 891-896.-   Non Patent Literature 2:-   Pharmaceutical and Medical Device Regulatory Science Project    supported by Health and Labour Sciences Research Grant “Atarashiku    Kaihatsu sareta Hib, Haien Kyukin, Rotavirus, HPV nado no Kaku    Wakuchin no Yukosei, Anzensei narabini sono Touyohouhou ni kansuru    Kisoteki Rinshoteki Kenkyu”, Shoni Shinshusei Kansensho Yurai Haien    Kyukin no Ekigakuteki Kaiseki. Keigo Shibayama, 46-53 March 2012-   Non Patent Literature 3:-   McDaniel L S, Ralph B A, McDaniel D O et al. 1994. Localization of    protection-eliciting epitopes on PspA of Streptococcus pneumoniae    between amino acid residues 192 and 260. Microb Pathog. 17: 323-37.-   Non Patent Literature 4:-   Daniels C C, Coan P, King J et al. 2010. The proline-rich region of    pneumococcal surface proteins A and C contains surface-accessible    epitopes common to all pneumococci and elicits antibody-mediated    protection against sepsis. Infect Immun. 78: 2163-2172.-   Non Patent Literature 5:-   Hollingshead S K, Becker R, Briles D E. 2000. Diversity of PspA:    mosaic genes and evidence for past recombination in Streptococcus    pneumoniae. Infect Immun. 68: 5889-5900.-   Non Patent Literature 6:-   Tu A H, Fulgham R L, McCrory M A et al. 1999. Pneumococcal surface    protein A inhibits complement activation by Streptococcus    pneumoniae. Infect Immun. 67: 4720-4724.-   Non Patent Literature 7:-   Ezoe H, Akeda Y, Piao Z, Aoshi T, Koyama S, Tanimoto T, Ken J. Ishii    K J, Oishi K. 2011. Intranasal vaccination with pneumococcal surface    protein A plus poly(I:C) protects against secondary pneumococcal    pneumonia in mice. Vaccine 29: 1754-1761.-   Non Patent Literature 8:-   Piao Z, Oma K, Ezoe H, Akeda Y, Tomono K, Oishi K. 2011. Comparative    effects of toll-like receptor agonists on a low dose PspA intranasal    vaccine against fatal pneumococcal pneumonia in mice. J Vaccines    Vaccin 2:1,    www.omicsonline.org/comparative-effects-of-toll-like-receptor-agonists-on-a-low-dose-pspa-intranasal-vaccine-against-fatal-pneumococcal-pneumonia-in-mice-2157-7560.1000113.pdf-   Non Patent Literature 9:-   Ren B, Szalai A J, Hollingshead S K et al. 2004. Effects of PspA and    antibodies to PspA on activation and deposition of complement on the    pneumococcal surface. Infect Immun. 72: 114-122.-   Non Patent Literature 10:-   Darrieux M, Moreno A T, Ferreira D M et al. 2008. Recognition of    pneumococcal isolates by antisera raised against PspA fragments    different clades. J Med Microbial. 57: 273-278. Non Patent    Literature 11:-   Moreno A T, Oliveira M L, Ferreira D M et al. 2010. Immunization of    mice with single PspA Fragments induces Antibodies capable of    mediating complement deposition on different pneumococcal strains    and cross-protection. Clin Vaccine Immunol. 17: 439-446.-   Non Patent Literature 12:-   M. Darrieux, E. N. Miyaji, D. M. Ferreira, L. M. Lopes, A. P. Y.    Lopes, B. Ren, D. E. Briles, S. K. Hollingshead, and L. C. C.    Leite. 2007. Fusion Proteins Containing Family 1 and Family 2 PspA    Fragments Elicit Protection against Streptococcus pneumoniae That    Correlates with Antibody-Mediated Enhancement of Complement    Deposition. Infect Immun. 75: 5930-5938.-   Non Patent Literature 13:-   Wei Xin, Yuhua Li, Hua Mo, Kenneth L. Roland, and Roy    Curtiss III. 2009. PspA Family Fusion Proteins Delivered by    Attenuated Salmonella enterica Serovar Typhimurium Extend and    Enhance Protection against Streptococcus pneumoniae. Infect Immun.    77: 4518-4528.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to identify a specific combinationof pneumococcal surface protein antigens PspAs of different clades orstrains, the combination having broadly cross-reactive immunogenicityand being capable of inducing immune response against a wide range ofpneumococcal clinical isolates, and to provide a novel pneumococcalvaccine based on such a combination of PspAs. In particular, an objectof the present invention is to identify a single protein antigen in theform of a fusion protein of a plurality of PspAs, the single proteinantigen having broadly cross-reactive immunogenicity and being capableof inducing immune response against a wide range of pneumococcalclinical isolates, and to provide a novel pneumococcal vaccine based onsuch a single protein antigen.

Solution to Problem

The present invention includes the following to achieve theabove-mentioned object.

[1] A pneumococcal vaccine comprising a fusion protein at leastcomprising a full-length family 1 pneumococcal surface protein A (PspA)(with the exception of PspAs of pneumococcal strains Rx1 and St435/96)or a fragment thereof, and a full-length family 2 PspA or a fragmentthereof.[2] The pneumococcal vaccine according to the above [1], wherein thefamily 1 PspA is a clade 2 PspA.[3] The pneumococcal vaccine according to the above [2], wherein thefusion protein is any one of the following (1) to (3):(1) a fusion protein at least comprising a family 1, clade 2 PspA and afamily 2, clade 3 PspA,(2) a fusion protein at least comprising a family 1, clade 2 PspA and afamily 2, clade 4 PspA, and(3) a fusion protein at least comprising a family 1, clade 2 PspA and afamily 2, clade 5 PspA.[4] The pneumococcal vaccine according to the above [3], wherein thefusion protein is any one of the following (4) to (6):(4) a fusion protein consisting of a family 1, clade 2 PspA and a family2, clade 3 PspA,(5) a fusion protein consisting of a family 1, clade 2 PspA and a family2, clade 4 PspA, and(6) a fusion protein consisting of a family 1, clade 2 PspA and a family2, clade 5 PspA.[5] The pneumococcal vaccine according to any one of the above [1] to[4], wherein the PspA fragment at least contains the whole or part of aproline-rich region.[6] The pneumococcal vaccine according to the above [5], wherein thePspA fragment consists of the whole or part of the proline-rich region,and the whole or part of an α-helical region adjacent thereto.[7] The pneumococcal vaccine according to the above [2], wherein thefamily 1, clade 2 PspA is from a pneumococcal strain selected from thegroup consisting of D39, WU2, E134, EF10197, EF6796, BG9163 and DBL5.[8] The pneumococcal vaccine according to the above [3], wherein thefamily 2, clade 3 PspA is from a pneumococcal strain TIGR4, BG8090 orAC122, the family 2, clade 4 PspA is from a pneumococcal strain EF5668,BG7561, BG7817 or BG11703, and the family 2, clade 5 PspA is from apneumococcal strain ATCC6303 or KK910.[9] The pneumococcal vaccine according to the above [1], wherein thefusion protein consists of an amino acid sequence which is identical oressentially identical to that represented by SEQ ID NO: 1, 3 or 5.[10] A pneumococcal vaccine at least comprising a full-length family 1,clade 2 PspA or a fragment thereof, and a full-length family 2 PspAselected from the group consisting of clade 3, 4 and 5 PspAs, or afragment thereof.[11] The pneumococcal vaccine according to the above [10], wherein thePspAs are any one of the following (i) to (iii):(i) a combination of only a family 1, clade 2 PspA and a family 2, clade3 PspA,(ii) a combination of only a family 1, clade 2 PspA and a family 2,clade 4 PspA, and(iii) a combination of only a family 1, clade 2 PspA and a family 2,clade 5 PspA.[12] The pneumococcal vaccine according to any one of the above [1] to[11], further comprising an adjuvant.[13] The pneumococcal vaccine according to any one of the above [1] to[12], further comprising a vaccine component against a pathogen otherthan pneumococci.

Advantageous Effects of Invention

The present invention can provide a pneumococcal vaccine that hasbroadly cross-reactive immunogenicity and can induce immune responseagainst a wide range of pneumococcal clinical isolates. The inoculationof the pneumococcal vaccine of the present invention can induceprotective immunity against pneumococcal infections in children andadults.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the structure of a PspA protein.

FIG. 2 shows the structures of three kinds of PspA-based fusion proteinsprepared in Example 1. (A) shows the structure of PspA2+4, (B) shows thestructure of PspA2+5, and (C) shows the structure of PspA3+2.

FIG. 3(A) shows the results of SDS-PAGE of the indicated PspA-basedfusion proteins, and FIG. 3(B) shows the results of western blotting ofthe indicated PspA-based fusion proteins.

FIG. 4 shows the results of the measurement of the capacities ofantiserum IgG to bind to the surface of pneumococcal cells of differentPspA clades (the X-axis shows test strains). The antisera were obtainedby immunization of mice with the indicated PspA-based fusion proteins.

FIG. 5 shows the results on the survival rates of PspA-based fusionprotein-immunized mice during 2 weeks after infection with variouspneumococci of different PspA clades. Immunization was performed usingthe indicated PspA-based fusion proteins. (A) shows the results ofinfection with 2×10⁷ CFU of BG9739, (B) shows the results of infectionwith 2×10⁷ CFU of WU2, (C) shows the results of infection with 5×10⁶ CFUof TIGR4, (D) shows the results of infection with 1×10⁸ CFU of KK1162,and (E) shows the results of infection with 5×10⁵ CFU of ATCC6303.

FIGS. 6A to 6C show the results of the measurement of the bindingcapacities of antiserum IgG for pneumococcal clinical isolates. Theantisera were obtained by immunization of mice with the indicatedPspA-based fusion proteins. FIG. 6A shows the results forPspA2+4-induced antiserum, FIG. 6B shows the results for PspA2+5-inducedantiserum, and FIG. 6C shows the results for PspA3+2-induced antiserum.

FIG. 7 shows the results of the measurement of anti-PspA antibody titersof the antisera obtained by immunization of mice with PspA2 alone, PspA3alone, a combination of PspA2 and PspA3, and PspA3+2 fusion protein.

FIG. 8 shows the results of the measurement of anti-PspA antibody titersof the antisera obtained by immunization of mice with three kinds ofPspA-based fusion proteins (PspA2+4, PspA2+5, PspA3+2) together with CpGalone or a combination of CpG and Alum as an adjuvant.

FIG. 9 shows the results of the measurement of anti-PspA antibody titersof the antisera obtained by immunization of mice with PspA3+2 fusionprotein together with various concentrations of Alum alone as anadjuvant.

DESCRIPTION OF EMBODIMENTS

<Pneumococcal Vaccine>

The present invention provides a pneumococcal vaccine comprising afusion protein at least comprising a full-length family 1 PspA or afragment thereof, and a full-length family 2 PspA or a fragment thereof.However, the present invention does not include a fusion proteincomprising a pneumococcal strain Rx1 PspA (family 1, clade 2), which isused for fusion proteins of a family 1, clade 2 PspA and a family 2,clade 4 PspA described in Non Patent Literature 13. Moreover, thepresent invention does not include a fusion protein comprising apneumococcal strain St435/96 PspA (family 1, clade 1) (for informationon the strain, see Non Patent Literature 12 and TABLE 1 of Miyaji E N etal., Infect Immun. 70: 5086-5090, 2002), which is used as a family 1,clade 1 PspA for fusion proteins described in Non Patent Literature 12.A partial sequence of the gene encoding the pneumococcal strain St435/96PspA, and the amino acid sequence thereof are registered with a databasesuch as GenBank under accession number AY082387. Hereinafter, it shouldbe noted that the “family 1 PspA” does not include the Rx1 PspA or theSt435/96 PspA.

In the pneumococcal vaccine of the present invention (hereinaftersometimes referred to as “the vaccine of the present invention”), thefusion protein (hereinafter sometimes referred to as “the fusion proteinof the present invention”) at least comprises a family 1 PspA and afamily 2 PspA. The fusion protein of the present invention may comprisethree or more kinds of PspAs, or comprise PspAs together with anotherprotein and/or a capsular polysaccharide (for example, a carrierprotein, a capsular antigen for vaccines, etc.). Preferably, the fusionprotein consists of two kinds of PspAs, i.e., a family 1 PspA and afamily 2 PspA. The fusion protein may comprise, in addition to the aminoacid sequences of PspAs, another amino acid sequence such as a tagsequence, a vector-derived sequence and a restriction enzyme sequence.

The order of the constituent proteins fused in the fusion protein of thepresent invention is not limited, and for example, in the case where thefusion protein is composed of two kinds of PspAs, i.e., family 1 andfamily 2 PspAs, the fusion protein may comprise the family 1 PspA at theN-terminal side and the family 2 PspA at the C-terminal side, oralternatively comprise the family 2 PspA at the N-terminal side and thefamily 1 PspA at the C-terminal side. Similarly, in the case where thefusion protein comprises three or more kinds of PspAs or comprises PspAstogether with another protein, the order of the constituent proteins inthe fusion protein is not limited.

For the fusion protein of the present invention, a PspA of apneumococcus of which the PspA family and clade are already identifiedcan preferably be used. In the case where a PspA of a pneumococcus ofwhich the PspA family and clade are unidentified is used, the PspAfamily and clade identification of the pneumococcus preferably precedesthe use. For the PspA family and clade identification, a PspA genesequence putatively containing an α-helical region and a proline-richregion is subjected to PCR amplification followed by gene sequencing,and an about 400-bp nucleotide sequence upstream of the proline-richregion in the sequenced gene is compared with the correspondingsequences in the PspA genes of which the clades are already identified.Specifically, when the about 400-bp nucleotide sequence has 97 to 100%homology to the corresponding sequence of any of the PspA genes shown inTables 1 and 2, both clades are regarded as the same. A PspA identifiedas clade 1 or 2 is defined as belonging to family 1, a PspA identifiedas clade 3, 4 or 5 is defined as belonging to family 2, and a PspAidentified as clade 6 is defined as belonging to family 3 (Reference:Non Patent Literature 5 and Swiatlo E, Brooks-Walter A, Briles D E,McDaniel L S. Oligonucleotides identity conserved and variable regionsof pspA and pspA-like sequences of Streptococcus pneumoniae. Gene 1997,188: 279-284). In an alternative identification method, a PspA gene ofinterest is amplified with a set of primers specific to family 1 or 2,and the length of the PCR product determines the family of the PspA.Specifically, when the length of the PCR product is about 1000 bp, thePspA is defined as belonging to family 1, and when the length of the PCRproduct is about 1200 bp, the PspA is defined as belonging to family 2(Reference: Vela Coral M C, Fonseca N, Castaneda E, Di Fabio J L,Hollingshead S K, Briles D E. Pneumococcal surface protein A of invasiveStreptococcus pneumoniae isolates from Colombian children. Emerg InfectDis 2001, 7: 832-6).

Exemplary pneumococci of which the PspA families and clades areidentified include pneumococcal strains shown in Tables 1 and 2, andPspAs of these pneumococcal strains (Rx1 excluded) can preferably beused for the fusion protein of the present invention.

TABLE 1 Family 1 Strain Serotype Clade GenBank Accession No. BG9739  4 1AF071804 DBL6A  6A 1 AF071805 L81905  4 1 AF071809 BG8743 23 1 AF071803AC94  9L 1 AF071802 BG6692 33 1 AF071808 BG8838  6 1 AF071807 DBL1  6B 1AF071806 Rx1 Rough 2 M74122 E134 23 2 AF071811 EF10197  3 2 AF071812EF6796  6A 2 AF071813 BG9163  6B 2 AF071815 DBL5  5 2 AF071810 WU2  3 2AF071814

TABLE 2 Family 2 GenBank Strain Serotype Clade Accession No. EF3296  4 3AF071816 BG8090 19 3 AF071817 AC122  9V 3 AF071818 EF5668  4 4 U89711BG7561 15 4 AF071824 BG7817 12 4 AF071826 BG11703 18 4 AF071821 ATCC6303 3 5 AF071820

In the fusion protein of the present invention, the family 1 PspA ispreferably a clade 2 PspA. The fusion protein is more preferably any ofthe following (1) to (3):

(1) a fusion protein at least comprising a family 1, clade 2 PspA and afamily 2, clade 3 PspA,

(2) a fusion protein at least comprising a family 1, clade 2 PspA and afamily 2, clade 4 PspA, and

(3) a fusion protein at least comprising a family 1, clade 2 PspA and afamily 2, clade 5 PspA.

The fusion protein is still more preferably any of the following (4) to(6):

(4) a fusion protein consisting of a family 1, clade 2 PspA and a family2, clade 3 PspA,

(5) a fusion protein consisting of a family 1, clade 2 PspA and a family2, clade 4 PspA, and

(6) a fusion protein consisting of a family 1, clade 2 PspA and a family2, clade 5 PspA.

The family 2 PspA is preferably a clade 3 PspA. Therefore, the vaccineof the present invention preferably comprises the above fusion protein(1) or (4).

The PspA used for the fusion protein may be a full-length PspA or afragment thereof. The two or more kinds of PspAs as the constituents ofthe fusion protein may be all full-length PspAs, a combination of afull-length PspA(s) and a PspA fragment(s), or all PspA fragments. APspA is first expressed as a protein (precursor) consisting of a signalsequence, an α-helical region, a proline-rich region, a choline-bindingregion and a C-terminal tail as shown in FIG. 1, and then the signalsequence is cleaved off, resulting in a mature PspA. That is, thefull-length PspA means a PspA having the same structure as FIG. 1 butlacking the signal sequence. The PspA fragment used for the fusionprotein of the present invention is not particularly limited as long asthe fragment consists of part of the full-length PspA and can induceprotective immunity against pneumococcal infections in a living body.Preferably, the PspA fragment contains the whole or part of theproline-rich region. In addition, the PspA fragment may further containthe whole or part of the α-helical region adjacent to the proline-richregion. Furthermore, it is preferable that the PspA fragment does notcontain the C-terminal tail, and it is more preferable that the PspAfragment does not contain the choline-binding region or the C-terminaltail. That is, the PspA fragment used for the fusion protein of thepresent invention is preferably a PspA fragment consisting of part ofthe proline-rich region; a PspA fragment consisting of the whole of theproline-rich region; a PspA fragment consisting of part of theproline-rich region and part of the α-helical region adjacent thereto; aPspA fragment consisting of part of the proline-rich region and thewhole of the α-helical region adjacent thereto; a PspA fragmentconsisting of the whole of the proline-rich region and part of theα-helical region adjacent thereto; or a PspA fragment consisting of thewhole of the proline-rich region and the whole of the α-helical region.More preferred is a PspA fragment consisting of the whole of theproline-rich region and the whole of the α-helical region.

The location of each region of a PspA can be determined according to thereport of Yother et al. (Yother J, Briles D E. 1992. Structuralproperties and evolutionary relationships of PspA, a surface protein ofStreptococcus pneumoniae, as revealed by sequence analysis. J.Bacteriol. 174: 601-609). Specifically, the α-helical region in a PspAof the Rx1 strain is a domain that is identical to an α-helix-containingregion (residues 1 to 288) predicted by a secondary structure predictionprogram but lacks the signal sequence (residues 1 to 31), and theα-helical region as used herein can be defined as a region having anamino acid sequence highly homologous to that of the above domain. Theproline-rich region can be defined as a region that is located betweenthe α-helical region and the choline-binding region and contains a highlevel of proline residues. The choline-binding region in a PspA of theRx1 strain is a domain having 10 repeats of a relatively highlyconserved 20-amino-acid sequence (based on TGWLQVNGSWYYLNANGAMA (SEQ IDNO: 24)), and the choline-binding region as used herein can be definedas a region having an amino acid sequence highly homologous to that ofthe above domain. The C-terminal tail can be defined as a region fromthe residue immediately following the final repeat in thecholine-binding region to the C-terminal stop codon.

The length of the PspA fragment is not particularly limited as long asthe length is sufficient for the induction of immune response in aliving body. The PspA fragment preferably consists of at least 27residues or more, more preferably 108 residues or more, and still morepreferably 300 residues or more (Reference: Daniels C C, Coan P, King J,Hale J, Benton K A, Briles D E, Hollingshead S K. The Proline-RichRegion of Pneumococcal Surface Proteins A and C ContainsSurface-Accessible Epitopes Common to All Pneumococci and ElicitsAntibody-Mediated Protection against Sepsis. Infect. Immun. 2010. 78:2163-2172).

Preferable examples of the family 1, clade 2 PspA include PspAs ofpneumococcal strains D39, WU2, E134, EF10197, EF6796, BG9163, DBL5, etc.More preferred are PspAs of D39 and WU2. Preferable examples of thefamily 2, clade 3 PspA include PspAs of pneumococcal strains TIGR4,BG8090, AC122, etc. More preferred is a PspA of TIGR4. Preferableexamples of the family 2, clade 4 PspA include PspAs of pneumococcalstrains EF5668, BG7561, BG7817, BG11703, etc. More preferred is a PspAof EF5668. Preferable examples of the family 2, clade 5 PspA includePspAs of pneumococcal strains ATCC6303, KK910, etc. More preferred is aPspA of ATCC6303.

The fusion protein of the present invention is preferably composed of acombination of a D39 PspA and an EF5668 PspA, a combination of a D39PspA and an ATCC6303 PspA, or a combination of a WU2 PspA and a TIGR4PspA. Among them, more preferred is a combination of a WU2 PspA and aTIGR4 PspA, and still more preferred is a combination of a TIGR4 PspA atthe N-terminal side and a WU2 PspA at the C-terminal side.

Furthermore, the fusion protein of the present invention preferablyconsists of an amino acid sequence which is identical or essentiallyidentical to that represented by SEQ ID NO: 1, 3 or 5. Among them, morepreferred is a fusion protein consisting of an amino acid sequence whichis identical or essentially identical to that represented by SEQ ID NO:5.

The amino acid sequence represented by SEQ ID NO: 1 constitutes a fusionprotein of a D39 PspA and an EF5668 PspA, in which a vector-derivedsequence containing a polyhistidine tag, a sequence of residues 32 to401 of the amino acid sequence of the D39 PspA (GenBank Accession No.ABJ54172, 619aa), a sequence corresponding to an EcoRI recognitionnucleotide sequence, and a sequence of residues 32 to 454 of the aminoacid sequence of the EF5668 PspA (GenBank Accession No. AAC62252, 653aa)are connected in this order from the N-terminus.

The amino acid sequence represented by SEQ ID NO: 3 constitutes a fusionprotein of a D39 PspA and an ATCC6303 PspA, in which a vector-derivedsequence containing a polyhistidine tag, a sequence of residues 32 to401 of the amino acid sequence of the D39 PspA (GenBank Accession No.ABJ54172, 619aa), a sequence corresponding to an EcoRI recognitionnucleotide sequence, and a sequence of residues 32 to 461 of a partialamino acid sequence of the ATCC6303 PspA (GenBank Accession No.AF071820, 461aa) are connected in this order from the N-terminus.

The amino acid sequence represented by SEQ ID NO: 5 constitutes a fusionprotein of a TIGR4 PspA and a WU2 PspA, in which a vector-derivedsequence containing a polyhistidine tag, a sequence of residues 32 to524 of the amino acid sequence of the TIGR4 PspA (Accession No.AAK74303, 744aa), a sequence corresponding to an EcoRI recognitionnucleotide sequence, and a sequence of residues 32 to 409 of a partialamino acid sequence of the WU2 PspA (Accession No. AAF27710, 415aa) areconnected in this order from the N-terminus.

The amino acid sequence essentially identical to that represented by SEQID NO: 1 is, for example, an amino acid sequence that is the same as SEQID NO: 1 except for having deletion, substitution or addition of one toseveral amino acids. As used herein, “deletion, substitution or additionof one to several amino acids” means deletion, substitution or additionof an amino acid (s) the number of which is practicable in a knownmethod for preparing mutant peptides, such as site-directed mutagenesis(preferably 10 or less amino acids, more preferably 7 or less aminoacids, and even more preferably 5 or less amino acids). Such a mutantprotein is not limited to a protein artificially mutated by a knownmethod for preparing mutant polypeptides, and may be a protein isolatedand purified from nature. In addition, the amino acid sequenceessentially identical to that represented by SEQ ID NO: 1 is, forexample, an amino acid sequence which is at least 80% identical, morepreferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identicalto that represented by SEQ ID NO: 1. The same holds for the amino acidsequence essentially identical to that represented by SEQ ID NO: 3 or 5.

The fusion protein consisting of the amino acid sequence essentiallyidentical to that represented by SEQ ID NO: 1 is preferably a fusionprotein having an activity of the essentially same nature as that of thefusion protein consisting of the amino acid sequence represented by SEQID NO: 1. The activity of the essentially same nature include theactivity of inducing immune response against a wide range ofpneumococcal strains, which is preferably at a level equivalent to (forexample, about 0.5- to 20-fold, preferably about 0.5- to 2-fold) that ofthe fusion protein consisting of the amino acid sequence represented bySEQ ID NO: 1. The same holds for the fusion protein consisting of theamino acid sequence essentially identical to that represented by SEQ IDNO: 3 or 5.

The fusion protein of the present invention can be prepared using knowngenetic engineering techniques, specifically by constructing arecombinant expression vector having an expressible insert of a geneencoding the fusion protein of the present invention, transfecting thevector into appropriate host cells for expression of a recombinantprotein, and purifying the recombinant protein. Alternatively, thepreparation of the fusion protein of the present invention can beperformed using a gene encoding the fusion protein of the presentinvention with a known in vitro coupled transcription-translation system(for example, a cell-free protein synthesis system derived from rabbitreticulocytes, wheat germ or Escherichia coli).

The vaccine of the present invention can induce immune response againstpneumococci without any adjuvant and thus is highly useful. However, thevaccine of the present invention may comprise one or more kinds ofadjuvants. In the case where the vaccine of the present inventioncomprises an adjuvant, the adjuvant can be selected as appropriate fromwell-known adjuvants. Specific examples of the well-known adjuvantsinclude aluminum adjuvants (for example, aluminum salts such as aluminumhydroxide, aluminum phosphate and aluminum sulfate, or any combinationthereof), complete or incomplete Freund's adjuvant, TLR ligands (forexample, CpG, Poly(I:C), Pam3CSK4, etc.), BAY, DC-chol, pcpp,monophosphoryl lipid A, QS-21, cholera toxin and formylmethionylpeptides. Preferable adjuvants are aluminum adjuvants, TLR ligands and acombination of both.

In the case where the vaccine of the present invention comprises anadjuvant, the amount of the adjuvant is not particularly limited as longas the amount is sufficient to nonspecifically enhance immune responseinduced by the fusion protein of the present invention, and the amountcan be selected as appropriate according to the kind of the adjuvantetc. For example, in the case where an aluminum adjuvant (aluminumhydroxide) and CpG are used in combination, it is preferable that thevaccine comprises an about 1- to 100-fold amount of the aluminumadjuvant and an about 1- to 50-fold amount of CpG relative to the amountof the fusion protein of the present invention on a mass basis.

The vaccine of the present invention may comprise a vaccine componentagainst a pathogen other than pneumococci. That is, the presentinvention provides a combination vaccine comprising the above-describedfusion protein of the present invention, which is a vaccine componentagainst pneumococci, and a vaccine component against a pathogen otherthan pneumococci. The vaccine component against a pathogen other thanpneumococci is not particularly limited, and for example, is a vaccinecomponent which has been practically used in combination vaccines.Specific examples of such a vaccine component include diphtheria toxoid,pertussis toxoid, Bordetella pertussis antigen, tetanus toxoid,inactivated poliovirus, attenuated measles virus, attenuated rubellavirus, attenuated mumps virus, Haemophilus influenzae type bpolysaccharide antigen, hepatitis B virus surface (HBs) antigen andinactivated hepatitis A virus antigen.

Examples of currently available combination vaccines includediphtheria-pertussis-tetanus vaccine (DPT vaccine),diphtheria-pertussis-tetanus-inactivated poliovirus vaccine (DPT-IPVvaccine), measles-rubella vaccine (MR vaccine), measles-mumps-rubellavaccine (MMR), Haemophilus influenzae type b (Hib)-hepatitis B virusvaccine, hepatitis A and B virus vaccine, anddiphtheria-pertussis-tetanus-hepatitis virus-inactivated poliovirusvaccine. It is preferable that any of these combination vaccines issupplemented with the fusion protein as a component of the pneumococcalvaccine of the present invention.

The vaccine of the present invention can be administered orally orparenterally. The parenteral administration includes intraperitonealadministration, subcutaneous administration, intracutaneousadministration, intramuscular administration, intravenousadministration, intranasal administration, transdermal administrationand transmucosal administration. Preferred is parenteral administrationand more preferred are intracutaneous administration, subcutaneousadministration and intramuscular administration.

For the formulation of the vaccine of the present invention, the fusionprotein of the present invention, a pharmaceutically acceptable carrierand if needed an additive are blended and formed into a dosage form.Specific examples of the dosage form include oral preparations such astablets, coated tablets, pills, powders, granules, capsules, solutions,suspensions and emulsions; and parenteral preparations such asinjections, infusions, suppositories, ointments and patches. Theblending ratio of the carrier or the additive can be determined asappropriate based on the usual range in the pharmaceutical field. Thecarrier or the additive that can be blended is not particularly limited,and the examples include various carriers such as water, physiologicalsaline, other aqueous solvents, and aqueous or oily bases; and variousadditives such as excipients, binders, pH adjusters, disintegrants,absorption enhancers, lubricants, colorants, corrigents and fragrances.

Examples of the additive used for solid oral preparations includeexcipients such as lactose, mannitol, glucose, microcrystallinecellulose and corn starch; binders such as hydroxypropyl cellulose,polyvinylpyrrolidone and magnesium aluminometasilicate; dispersants suchas corn starch; disintegrants such as calcium carboxymethyl cellulose;lubricants such as magnesium stearate; solubilizing agents such asglutamic acid and aspartic acid; stabilizers; water soluble polymersincluding celluloses such as hydroxypropyl cellulose,hydroxypropylmethyl cellulose and methyl cellulose, and syntheticpolymers such as polyethylene glycol, polyvinylpyrrolidone and polyvinylalcohol; sweeteners such as white sugar, powder sugar, sucrose,fructose, glucose, lactose, reduced malt sugar syrup (maltitol syrup),reduced malt sugar syrup powder (maltitol syrup powder), high-glucosecorn syrup, high-fructose corn syrup, honey, sorbitol, maltitol,mannitol, xylitol, erythritol, aspartame, saccharin and saccharinsodium; and coating agents such as white sugar, gelatin, hydroxypropylcellulose and hydroxypropylmethyl cellulose phthalate.

The formulation of liquid oral preparations involves dissolution,suspension or emulsification in a generally used diluent. Examples ofthe diluent include purified water, ethanol and a mixture thereof. Theliquid oral preparation may further contain a wetting agent, asuspending agent, an emulsifier, a sweetener, a flavoring agent, afragrance, a preservative, a buffering agent and/or the like.

Examples of the additive used for injections for oral administrationinclude isotonizing agents such as sodium chloride, potassium chloride,glycerin, mannitol, sorbitol, boric acid, borax, glucose and propyleneglycol; buffering agents such as a phosphate buffer solution, an acetatebuffer solution, a borate buffer solution, a carbonate buffer solution,a citrate buffer solution, a Tris buffer solution, a glutamate buffersolution and an ϵ-aminocaproate buffer solution; preservatives such asmethyl parahydroxybenzoate, ethyl parahydroxybenzoate, propylparahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzylalcohol, benzalkonium chloride, sodium dehydroacetate, disodium edetate,boric acid and borax; thickeners such as hydroxyethyl cellulose,hydroxypropyl cellulose, polyvinyl alcohol and polyethylene glycol;stabilizers such as sodium hydrogen sulfite, sodium thiosulfate,disodium edetate, sodium citrate, ascorbic acid anddibutylhydroxytoluene; and pH adjusters such as hydrochloric acid,sodium hydroxide, phosphoric acid and acetic acid. The injection mayfurther contain an appropriate solubilizer. Examples of the solubilizerinclude alcohols such as ethanol; polyalcohols such as propylene glycoland polyethylene glycol; and nonionic surfactants such as polysorbate80, polyoxyethylene hydrogenated castor oil 50, lysolecithin andPluronic polyol. Liquid preparations such as injections can be directlypreserved by freezing or preserved after removal of water bylyophilization etc. Lyophilized preparations can be reconstituted indistilled water for injection or the like just before use.

The vaccine of the present invention can be administered to any animal(a human or a non-human animal) that has an immune system. Examples ofthe animal include mammals such as humans, monkeys, cattle, horses,pigs, sheep, goats, dogs, cats, guinea pigs, rats and mice; and birdssuch as chickens, ducks and geese. Preferably, the vaccine of thepresent invention is administered to a human child or adult.

In the administration of the vaccine of the present invention, thedosing frequency and interval are not particularly limited. For example,the vaccine may be administered once, or multiple times at intervals ofabout two days to about eight weeks.

Although the dose of the vaccine varies with the administration subject,the administration method, etc., the dose per administration ispreferably about 0.01 μg to about 10 mg, more preferably about 0.1 μg toabout 1 mg, and still more preferably about 1 μg to about 0.1 mg.

The present invention includes a method for prevention or treatment ofpneumococcal infections, and the method comprises administering aneffective amount of the vaccine of the present invention to an animal.

The vaccine of the present invention has the following advantages overconventional pneumococcal vaccines which are conjugate vaccines usingcapsular polysaccharides as antigens.

1) The vaccine is effective against a wide variety of pneumococcalstrains despite using a single fusion protein antigen.

2) Since the vaccine uses a protein antigen, the vaccine can be producedwithout the step of fusion with a carrier protein, and thus theproduction cost is low.

3) Since the vaccine uses a protein antigen, the vaccine does notrequire any carrier protein for induction of protective immunity in bothchildren and adults.

4) The vaccine uses a single fusion protein and there is no need to mixa plurality of antigens.

5) The vaccine can be produced by a simple process involvingpurification of just one kind of fusion protein (vaccine antigen), andthus the production cost can be reduced.

The present invention include a pneumococcal vaccine at least comprisinga full-length family 1, clade 2 PspA or a fragment thereof, and afull-length family 2 PspA selected from the group consisting of clade 3,4 and 5 PspAs, or a fragment thereof. That is, the present inventioninclude a pneumococcal vaccine at least comprising a clade 2 PspA and aclade 3 PspA, a pneumococcal vaccine at least comprising a clade 2 PspAand a clade 4 PspA, and a pneumococcal vaccine at least comprising aclade 2 PspA and a clade 5 PspA. In these embodiments of thepneumococcal vaccine, the PspAs in each of these three combinations maybe present as separate proteins. Except for this point, theseembodiments of the pneumococcal vaccine can be practiced in the samemanner as the above-described pneumococcal vaccine comprising the fusionprotein of the present invention.

In the case where the PspAs in each of the above-described threecombinations are present as separate proteins in the pneumococcalvaccine, the PspAs are preferably any of the following (i) to (iii):

(i) a combination of only a family 1, clade 2 PspA and a family 2, clade3 PspA,

(ii) a combination of only a family 1, clade 2 PspA and a family 2,clade 4 PspA, and

(iii) a combination of only a family 1, clade 2 PspA and a family 2,clade 5 PspA.

In the case where the PspAs in each of the above-described threecombinations are present as separate proteins, the PspA fragment usedpreferably contains the same regions as those contained in the PspAfragment used for the above-described fusion protein. Preferable kindsof PspA-expressing pneumococcal strains are the same as those describedfor the fusion protein. Specifically, the clade 2 PspA is preferably aprotein consisting of an amino acid sequence which is identical oressentially identical to that represented by SEQ ID NO: 25 or 26. Theclade 3 PspA is preferably a protein consisting of an amino acidsequence which is identical or essentially identical to that representedby SEQ ID NO: 27. The clade 4 PspA is preferably a protein consisting ofan amino acid sequence which is identical or essentially identical tothat represented by SEQ ID NO: 28. The clade 5 PspA is preferably aprotein consisting of an amino acid sequence which is identical oressentially identical to that represented by SEQ ID NO: 29.

The amino acid sequence represented by SEQ ID NO: 25 constitutes aprotein consisting of the full-length α-helical region and thefull-length proline-rich region of a D39 PspA (the amino acid sequenceis shown in SEQ ID NO: 30, and the nucleotide sequence of thecorresponding gene is shown in SEQ ID NO: 31), and corresponds toresidues 32 to 401 of the amino acid sequence of the D39 PspA (SEQ IDNO: 30).

The amino acid sequence represented by SEQ ID NO: 26 constitutes aprotein consisting of the full-length α-helical region and thefull-length proline-rich region of a WU2 PspA (its partial amino acidsequence is shown in SEQ ID NO: 32, and the nucleotide sequence of thecorresponding gene is shown in SEQ ID NO: 33), and corresponds toresidues 32 to 409 of the partial amino acid sequence of the WU2 PspA(SEQ ID NO: 32).

The amino acid sequence represented by SEQ ID NO: 27 constitutes aprotein consisting of the full-length α-helical region and thefull-length proline-rich region of a TIGR4 PspA (the amino acid sequenceis shown in SEQ ID NO: 34, and the nucleotide sequence of thecorresponding gene is shown in SEQ ID NO: 35), and corresponds toresidues 32 to 524 of the amino acid sequence of the TIGR4 PspA (SEQ IDNO: 34).

The amino acid sequence represented by SEQ ID NO: 28 constitutes aprotein consisting of the full-length α-helical region and thefull-length proline-rich region of an EF5668 PspA (the amino acidsequence is shown in SEQ ID NO: 36, and the nucleotide sequence of thecorresponding gene is shown in SEQ ID NO: 37), and corresponds toresidues 32 to 454 of the amino acid sequence of the EF5668 PspA (SEQ IDNO: 36).

The amino acid sequence represented by SEQ ID NO: 29 constitutes aprotein consisting of the full-length α-helical region and thefull-length proline-rich region of an ATCC6303 PspA (its partial aminoacid sequence is shown in SEQ ID NO: 38, and the nucleotide sequence ofthe corresponding gene is shown in SEQ ID NO: 39), and corresponds toresidues 32 to 461 of the amino acid sequence of the ATCC6303 PspA (SEQID NO: 38).

The protein consisting of an amino acid sequence essentially identicalto that represented by any of SEQ ID NOS: 25 to 29 is defined in thesame manner as set forth above regarding the protein consisting of theamino acid sequence essentially identical to the amino acid sequencerepresented by SEQ ID NO: 1.

<Polynucleotide>

The present invention provides a polynucleotide encoding the fusionprotein of the present invention. The polynucleotide can be present inthe form of RNA (for example, mRNA) or DNA (for example, cDNA or genomicDNA). The polynucleotide may be a double or single strand. The doublestrand may be a double-stranded DNA, a double-stranded RNA or a DNA-RNAhybrid. The single strand may be a coding strand (sense strand) or anon-coding strand (antisense strand). The polynucleotide of the presentinvention may be fused with a polynucleotide encoding a tag for labeling(a tag sequence or a marker sequence) at the 5′- or 3′-terminus. Thepolynucleotide of the present invention may further contain anuntranslated region (UTR) sequence, a vector sequence (including anexpression vector sequence), etc.

The polynucleotide of the present invention can be produced by obtainingtwo or more polynucleotides encoding different constituent PspAs of thefusion protein, and joining them. The polynucleotide encoding eachconstituent PspA of the fusion protein can be obtained by a well-knownDNA synthesis method, PCR, etc. To be more specific, in one example,based on the amino acid sequence of each constituent PspA of the fusionprotein of the present invention, the nucleotide sequence is designed byappropriate selection of a codon for each amino acid, and the designednucleotide sequence is chemically synthesized on a commercial DNAsynthesizer to give a desired polynucleotide. In another example, theinformation on the nucleotide sequence of the gene encoding a PspA ofinterest is obtained from a well-known database (GenBank etc.) (see theaccession numbers in Tables 1 and 2), primers for amplifying a desiredregion of the PspA-encoding gene are designed based on the information,and using these primers, PCR amplification from the genomic DNA of apneumococcus expressing the PspA of interest is performed to give adesired DNA fragment in a large amount. The thus-prepared separatepolynucleotides encoding different constituent PspAs of the fusionprotein are joined using a genetic engineering technique to give thepolynucleotide of the present invention.

The polynucleotide encoding a fusion protein consisting of the aminoacid sequence represented by SEQ ID NO: 1 is, for example, apolynucleotide consisting of the nucleotide sequence represented by SEQID NO: 2, but is not limited thereto. The polynucleotide encoding afusion protein consisting of the amino acid sequence represented by SEQID NO: 3 is, for example, a polynucleotide consisting of the nucleotidesequence represented by SEQ ID NO: 4, but is not limited thereto. Thepolynucleotide encoding a fusion protein consisting of the amino acidsequence represented by SEQ ID NO: 5 is, for example, a polynucleotideconsisting of the nucleotide sequence represented by SEQ ID NO: 6, butis not limited thereto.

<Expression Vector>

The present invention provides an expression vector used for theproduction of the fusion protein of the present invention. Theexpression vector of the present invention is not particularly limitedas long as it contains a polynucleotide encoding the fusion protein ofthe present invention, but preferred are plasmid vectors carrying arecognition sequence for RNA polymerase (pSP64, pBluescript, etc.). Themethod for preparing the expression vector is not particularly limited,and the expression vector may be prepared with the use of a plasmid, aphage, a cosmid or the like. The kind of the vector is not particularlylimited and any appropriate vector that can be expressed in host cellscan be selected. For example, depending on the kind of the host cell, anappropriate promoter sequence to ensure the expression of thepolynucleotide of the present invention is selected, and this promotersequence and the polynucleotide of the present invention are insertedinto a plasmid etc. to give a desired expression vector. After a hosttransformed with the expression vector of the present invention iscultured, cultivated or bred, the fusion protein of the presentinvention can be collected and purified from the culture products etc.by conventional methods (for example, filtration, centrifugation, celldisruption, gel filtration chromatography, ion exchange chromatography,affinity chromatography, etc.).

The expression vector preferably contains at least one selection marker.Examples of the marker include a dihydrofolate reductase gene and aneomycin resistance gene for eukaryote cell culture; and a tetracyclineresistance gene, an ampicillin resistance gene and a kanamycinresistance gene for culture of Escherichia coli (E. coli) and otherbacteria. Such a selection marker is useful for checking whether thepolynucleotide of the present invention has been successfullytransfected into host cells and is reliably expressed therein.

The host cell is not particularly limited and various known cells canpreferably be used. Specific examples of the host cell include bacteriasuch as E. coli, yeasts (budding yeast Saccharomyces cerevisiae andfission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditiselegans), Xenopus laevis oocytes and animal cells (for example, CHOcells, COS cells and Bowes melanoma cells). The method for transfectingthe expression vector into host cells, i.e. the transformation method,is also not particularly limited and known methods such aselectroporation, the calcium phosphate method, the liposome method andthe DEAE dextran method can preferably be used.

<Transformant>

The present invention provides a transformant carrying the expressionvector of the present invention. As used herein, the transformantencompasses a cell, a tissue and an organ as well as an individualorganism. The kind of the organism to be transformed is not particularlylimited, and the examples include various microorganisms, plants andanimals listed above as examples of the host cell. The transformant ofthe present invention can preferably be used for the production of thefusion protein of the present invention. It is preferable that thetransformant of the present invention stably expresses the fusionprotein of the present invention, but a transformant transientlyexpressing the same can also be used.

The present invention also include the following.

[1] A fusion protein at least comprising a full-length family 1 PspA ora fragment thereof, and a full-length family 2 PspA or a fragmentthereof.

[2] The fusion protein according to the above [1], being any one of thefollowing (1) to (3):

(1) a fusion protein consisting of a family 1, clade 2 PspA and a family2, clade 3 PspA,

(2) a fusion protein consisting of a family 1, clade 2 PspA and a family2, clade 4 PspA, and

(3) a fusion protein consisting of a family 1, clade 2 PspA and a family2, clade 5 PspA.

[3] The fusion protein according to the above [1] or [2], wherein thefamily 2 PspA is a clade 3 PspA.

[4] The fusion protein according to the above [1], wherein the PspAfragment at least contains the whole or part of a proline-rich region.

[5] The fusion protein according to the above [4], wherein the PspAfragment consists of the whole or part of the proline-rich region, andthe whole or part of an α-helical region adjacent thereto.

[6] The fusion protein according to the above [1], consisting of anamino acid sequence which is identical or essentially identical to thatrepresented by SEQ ID NO: 1, 3 or 5.

[7] A polynucleotide encoding the fusion protein according to any one ofthe above [1] to [6].

[8] An expression vector containing the polynucleotide according to theabove [7].

[9] A transformant carrying the expression vector according to the above[8].

[10] Use of the fusion protein according to any one of the above [1] to[6] for production of a pneumococcal vaccine.

[11] A pneumococcal vaccine comprising the fusion protein according toany one of the above [1] to [6].

[12] The pneumococcal vaccine according to the above [11], furthercomprising an adjuvant.

[13] A method for prevention or treatment of pneumococcal infections,comprising administering an effective amount of the fusion proteinaccording to any one of the above [1] to [6] to an animal.

[14] The fusion protein according to any one of the above [1] to [6] foruse for prevention or treatment of pneumococcal infections.

EXAMPLES

Hereinafter, the present invention will be illustrated in detail byexamples, but is not limited thereto.

Example 1: Preparation of PspA-Based Fusion Proteins

The three kinds of PspA-based fusion proteins shown in (A) (B) and (C)of FIG. 2 were prepared with the use of the PspAs of D39, TIGR4, EF5668,ATCC6303 and WU2 among the pneumococcal strains shown in Table 3. Allgene cloning procedures were performed in E. coli DH5α. E. coli DH5α wascultured in LB medium (1% bacto tryptone, 0.5% yeast extract and 0.5%NaCl) and as needed, kanamycin was added at a final concentration of 30μg/ml in the medium.

TABLE 3 PspA GenBank Strain Serotype clade Origin Accession No. BG9739 4 1 UAB AF071804 D39  2 2 UAB CP000410.1 WU2  3 2 UAB AF071814 TIGR4  33 UAB AE005672.3 EF5668  4 4 UAB U89711 KK1162 11A 4 This study ATCC6303 3 5 UAB AF071820 BG6380 37 6 UAB AF071823 UAB: University of Alabama atBirmingham, USA.

A DNA fragment encoding the N-terminal α-helical and proline-richregions of the PspA of each strain was PCR-amplified from the genomicDNA of each strain with a specific set of primers shown in Table 4, anda desired pair of the PCR products were inserted into a pET28a(+) vectorto give an expression vector for a PspA-based fusion protein. Thespecific procedure is described below.

TABLE 4 Nucleotide Sequence (Bold letters indicate SEQ ID Primerrestriction enzyme recognition site) NO.  P1 (NdeI-D39)GGAATTCCATATGGAAGAATCTCCCGTAGCCAGT  7  P2 (D39-EcoRI)GGAATTCTTTTGGTGCAGGAGCTGG  8  P3 (EcoRI-EF5668)GGAATTCGAAGAATCTCCCGTAGCTAG  9  P4 (EF5668-XhoI)CCGCTCGAGTTAGTGCAAGGAGCTGGTTTG 10  P5 (EcoRI-ATCC6303)GGAATTCGAAGAATCTCCACAAGTTGTCG 11  P6 (ATCC6303-XhoI)CCGCTCGAGTTATGGTGCAGGAACTGGTTG 12  P7 (NdeI-TIGR4)GGAATTCCATATGGAAGAATCTCCACAAGTTGTC 13  P8 (TIGR4-EcoRI)GGAATTCTGGAGTGGCTGGTTTTTCTG 14  P9 (EcoRI-WU2)GGAATTCGAAGAATCTCCCGTAGCTAG 15 P10 (WU2-XhoI)CCGCTCGAGTTACTCTGGTTGTGGTGCAGGAGCTGGTTT 16 P11 (NdeI-BG9739)GGAATTCCATATGGAAGAAGCCCCCGTAGCTAG 19 P12 (BG9739-XhoI)CCGCTCGAGTTATTCTGGTTTAGGAGCTGGAG 20 P13 (D39-XhoI)CCGCTCGAGTTATTTTGGTGCAGGAGCTGG 21 P14 (TIGR4-XhoI)CCGCTCGAGTTATGGAGTGGCTGGTTTTTCTG 22 P15 (NdeI-EF5668)GGAATTCCATATGGAAGAATCTCCCGTAGCTAG 23

PCR amplification of a pneumococcal strain D39 PspA gene (with primersP1 and P2) and PCR amplification of a pneumococcal strain TIGR4 PspAgene (with primers P7 and P8) gave products with 5′-end NdeI and 3′-endEcoRI restriction enzyme recognition sequences, and these PCR productswere separately inserted into a pET28a(+) vector between the NdeI andEcoRI restriction sites. Next, PCR amplification of a pneumococcalstrain EF5668 PspA gene (with primers P3 and P4) and PCR amplificationof a pneumococcal strain ATCC6303 PspA gene (with primers P5 and P6)gave products with 5′-end EcoRI and 3′-end XhoI restriction enzymerecognition sequences, and these PCR products were separately insertedbetween the EcoRI and XhoI restriction sites of the above-preparedpET28a(+) vector having a D39 PspA gene insert, to give two kinds ofexpression vectors, i.e., expression vectors for D39- and EF5668-derivedPspA-based fusion protein PspA2+4 and for D39- and ATCC6303-derivedPspA-based fusion protein PspA2+5. In addition, PCR amplification of apneumococcal strain WU2 PspA gene (with primers P9 and P10) gave aproduct with 5′-end EcoRI and 3′-end XhoI restriction enzyme recognitionsequences, and this PCR product was inserted between the EcoRI and XhoIrestriction sites of the above-prepared pET28a(+) vector having aPCR-amplified insert of the TIGR4 PspA gene, to give an expressionvector for TIGR4- and WU2-derived PspA-based fusion protein PspA3+2. Thenucleotide sequences of these three fusion genes (PspA2+4 gene, PspA2+5gene and PspA3+2 gene) were verified with a DNA sequencer and identifiedas the nucleotide sequences represented by SEQ ID NOS: 2, 4 and 5,respectively.

The obtained PspA-based fusion protein expression vectors wereseparately used to transform E. coli BL21 (DE3), and the resulting threedifferent transformants were cultured at 37° C. with shaking in an LBmedium supplemented with 30 μg/ml kanamycin. For each transformant, whenthe absorbance at 600 nm (OD600) of the culture medium became about 0.8,IPTG (final concentration 0.5 mM) was added thereto, and then shakingculture was continued for another 3 hours to allow the expression of thePspA-based fusion protein in a large amount. After the cells werecollected, the PspA-based fusion protein was extracted therefrom andpurified by Ni²⁺ affinity chromatography using a polyhistidine tagattached to the fusion protein at the N-terminus, followed by gelfiltration. The purified fusion protein was subjected to SDS-PAGE (seeFIG. 3A) and subsequent western blotting with an anti-PspA antibody (seeFIG. 3B), and identified as a desired fusion protein.

Example 2: Capacities of PspA-Based Fusion Protein-Induced Antiserum IgGto Bind to the Surface of Pneumococcal Cells of Different PspA Clades

(1) Immunization of Mice with PspA-Based Fusion Proteins

A given PspA-based fusion protein (0.1 μg of PspA2+4, PspA2+5 orPspA3+2) and an adjuvant (2.5 μg of CpGK3 and 5.0 μg of Alum) were mixedin LPS-free PBS, and subcutaneously injected into female 6-week-oldC57/BL6j mice for vaccination. Each immunized group consists of 5 mice.The vaccination was performed every week, 3 times in total. One weekafter the final immunization (3rd vaccination), the blood was drawn fromeach mouse and the serum was separated.

(2) Measurement of Capacities of Antiserum IgG to Bind to the Surface ofPneumococcal Cells

Six pneumococcal strains corresponding to PspA clades 1 to 6 were used.Specifically, BG9739 was used as the strain of PspA clade 1, WU2 wasused as the strain of PspA clade 2, TIGR4 was used as the strain of PspAclade 3, KK1162 was used as the strain of PspA clade 4, ATCC6303 wasused as the strain of PspA clade 5, and BG6380 was used as the strain ofPspA clade 6 (see Table 3). Each pneumococcal strain was cultured in THYmedium (Todd-Hewitt broth supplemented with 0.5% yeast extract). Duringthe logarithmic growth phase, glycerol was added at a finalconcentration of 25% in the culture medium and the strain wascryopreserved at −80° C. before use.

The pneumococcal strain was cultured on a blood agar medium overnight,subcultured on a fresh blood agar medium for 4 to 5 hours, and thencollected in PBS. Ninety microliters of a pneumococcal suspensioncontaining about 10⁷ CFU was reacted with 10 μl of an antiserum (amixture of antisera from the animals in the same immunized group) at 37°C. for 30 minutes. The reaction mixture was further reacted with anFITC-labeled anti-mouse IgG goat antibody, subsequent washing andcentrifugation were performed, and the fluorescence intensity on thecells was measured by flow cytometry.

(3) Results

The results are shown in FIG. 4. The binding level is expressed as apercentage of the number of antiserum IgG-bound pneumococcal cells in10000 pneumococcal cells. The PspA3+2-induced antiserum showed highbinding to any of the pneumococcal strains of PspA clades 1 to 5. ThePspA2+4-induced antiserum and the PspA2+5-induced antiserum showed aslightly low binding to TIGR4, a pneumococcal strain of PspA clade 3,but showed high binding to the pneumococcal strains of the other PspAclades. Regarding the binding to BG6380, a pneumococcal strain of PspAclade 6, the PspA2+5-induced antiserum showed the highest value.

Example 3: Infection Protective Effect of Immunization with PspA-BasedFusion Proteins in Fatal Pneumonia Murine Model

(1) Experimental Method

Mice were immunized with the PspA-based fusion proteins in the samemanner as in Example 2. Mice injected with an adjuvant alone wereassigned to a negative control. The below-mentioned pneumococcal strainsexpressing PspAs of clades 1 to 5 (see Table 3) were transnasallyinjected into the mice 2 weeks after the final immunization (3rdvaccination), to create fatal pneumonia murine models. The lethalinfection doses per mouse were 2×10⁷ CFU for BG9739 (clade 1), 2×10⁷ CFUfor WU2 (clade 2), 5×10⁶ CFU for TIGR4 (clade 3), 1×10⁸ CFU for KK1162(clade 4), and 5×10⁵ CFU for ATCC6303 (clade 5). The number of animalsper group was 10, or in some cases, 8 (BG9739-infected PspA2+5-immunizedgroup, KK1162-infected negative control group, KK1162-infectedPspA3+2-immunized group, and ATCC6303-infected negative control group).

The survival rate was monitored over 2 weeks after the pneumococcalinfection of the immunized mice. The differences in the survival ratesamong the groups were analyzed by the Kaplan-Meier log-rank test. Whenthe P value was smaller than 0.05, the difference was regarded asstatistically significant.

(2) Results

The results are shown in FIG. 5. In FIG. 5, the symbol * indicatessignificant difference at P<0.05 in the mouse survival rates between thevaccination group and the adjuvant injection group, and the symbol **indicates significant difference at P<0.01 in the same comparison. (A)shows the results for BG9739 (clade 1), (B) shows the results for WU2(clade 2), (C) shows the results for TIGR4 (clade 3), (D) shows theresults for KK1162 (clade 4), and (E) shows the results for ATCC6303(clade 5). In the PspA3+2-immunized mice, the survival rate afterinfection with any of the pneumococcal strains of PspA clades 1 to 5 wassignificantly improved. In both the PspA2+4-immunized mice and thePspA2+5-immunized mice, the survival rate after infection with thepneumococcal strain of PspA clade 2, 4 or 5 was significantly improved.

Example 4: Measurement of Binding Capacities of Antiserum IgG forPneumococcal Clinical Isolates

(1) Experimental Method

Using the PspA-based fusion protein-induced antisera obtained in Example2, the binding capacities of antiserum IgG for pneumococcal clinicalisolates were measured. The procedure was the same as that in Example 2except that the pneumococcal strains used were clinical isolates.

The PspA families and clades of the pneumococcal clinical isolates wereidentified as follows. PCR amplification from the genomic DNA of eachpneumococcal clinical isolate was performed with the primers LSM12 andSKH2 shown below, and the PCR product was sequenced. An about 400-bpnucleotide sequence upstream of the proline-rich region in the sequencedgene was compared with the corresponding sequences in the PspA genes ofwhich the families and clades were already identified, and thereby thefamily and clade of each pneumococcal clinical isolate were determined(Reference: Pimenta F C, Ribeiro-Dias F, Brandileone M C et al. 2006.Genetic diversity of PspA types among nasopharyngeal isolates collectedduring an ongoing surveillance study of children in Brazil. J ClinMicrobiol. 44: 2838-43).

LSM12:  (SEQ ID NO: 17) CCGGATCCAGCGTCGCTATCTTAGGGGCTGGTT  SKH2: (SEQ ID NO: 18) CCACATACCGTTTTCTTGTTTCCAGCC (2) Evaluation Criterion and Results

Since the binding of antiserum IgG to PspA proteins on the pneumococcalcell surface is essential for infection protective effect as describedabove, the binding capacity of IgG for pneumococcal cells was measuredand used for the evaluation of the coverage of pneumococcal clinicalisolates by each PspA-based fusion protein.

The results are shown in FIG. 6. In FIG. 6, the serotype and PspA cladeof each pneumococcal clinical isolate are shown in the parentheses. In(A) PspA2+4-induced antiserum, (B) PspA2+5-induced antiserum and (C)PspA3+2-induced antiserum, the binding capacities of antiserum IgG foralmost all the pneumococcal clinical isolates (97.0%) met the criterion(binding level of 10% or more).

Example 5: Measurement of Anti-PspA Antibody Titers of PspAProtein-Induced Antisera

(1) Preparation of Antigen Proteins of Different PspA Clades

For each of clades 1 to 5, a recombinant PspA consisting of an α-helicalregion and a proline-rich region was prepared and used as an antigenprotein. The clade 1 PspA used was from BG9739, the clade 2 PspA usedwas from D39, the clade 3 PspA used was from TIGR4, the clade 4 PspAused was from EF5668, and the clade 5 PspA used was from ATCC6303.Expression vectors for the antigen proteins of different PspA cladeswere prepared as follows.

For each PspA, a DNA fragment encoding α-helical and proline-richregions was PCR-amplified to give a product with 5′-end NdeI and 3′-endXhoI restriction enzyme recognition sequences. The PCR primers used wereprimers P11 and P12 for BG9739, primers P1 and P13 for D39, primers P7and P14 for TIGR4, primers P15 and P4 for EF5668, and primers P7 and P6for ATCC6303. The obtained PCR products were separately inserted into apET28a(+) vector between the NdeI and XhoI restriction sites. Theobtained expression vectors were separately used to transform E. coliBL21 (DE3), and the resulting five different transformants were culturedat 37° C. with shaking in an LB medium supplemented with 30 μg/mlkanamycin. For each transformant, when the OD600 of the culture mediumbecame about 0.8, IPTG (final concentration 0.5 mM) was added thereto,and then shaking culture was continued for another 3 hours to allow theexpression of the PspA-based antigen protein in a large amount. Afterthe cells were collected, the PspA-based antigen protein was extractedtherefrom and purified by Ni²⁺ affinity chromatography using apolyhistidine tag attached to the antigen protein at the N-terminus,followed by gel filtration. The purified PspA-based antigen protein wassubjected to SDS-PAGE and subsequent western blotting, and identified asa desired protein.

(2) Immunization of Mice with Antigens

The antigens used were the above-prepared clade 2 PspA-based antigenprotein (PspA2) and clade 3 PspA-based antigen protein (PspA3), and aPspA-based fusion protein prepared in Example 1 (PspA3+2). Female6-week-old C57/BL6j mice were divided into the following five groupseach consisting of 5 mice.

-   -   PspA3+PspA2-immunized group (0.05 μg of PspA3, 0.05 μg of PspA2        and an adjuvant were injected)    -   PspA3+2-immunized group (0.1 μg of PspA3+2 and an adjuvant were        injected)    -   PspA2-immunized group (0.1 μg of PspA2 and an adjuvant were        injected)    -   PspA3-immunized group (0.1 μg of PspA3 and an adjuvant were        injected)    -   PspA3+2-immunized group (4.0 μg of PspA3+2 was injected)

The adjuvant used was 2.5 μg of CpGK3 and 5.0 μg of Alum. An antigensolution was prepared in LPS-free PBS and subcutaneously injected intothe mice for vaccination. The vaccination was performed every week, 3times in total. One week after the final immunization (3rd vaccination),the blood was drawn from each mouse and the serum was separated.

(3) ELISA

The purified antigen protein of each PspA clade was prepared at 5 μg/ml,and added to 96-well plates at 100 μl/well. The plates were allowed tostand at 4° C. overnight to give antigen-coated plates. The plates werewashed with PBST (PBS containing 0.05% Tween 20), serially dilutedsamples of each antiserum were added at 50 μl/well, and the plates wereallowed to stand at 37° C. for 30 minutes. After this, each well waswashed with PBST, a 2000-fold diluted alkaline phosphatase-labeledanti-mouse IgG goat antibody was added at 100 μl/well, and the plateswere allowed to stand at room temperature with protection from light for45 minutes. Subsequently, the absorbance at 405 nm (OD405) was measured.The anti-PspA antibody titer of each antiserum was expressed as the Log₂of the reciprocal of the antiserum dilution at which the absorbance was0.1 after subtraction of the absorbance of the negative control was 0.1.

(4) Results

The results are shown in FIG. 7. When PspA2 was used alone, the antibodytiter against clade 3 PspA was low, and when PspA3 was used alone, theantibody titers against the PspAs of clades 1, 2, 4 and 5 were low. WhenPspA2 and PspA3 were used in combination, the antibody titers againstall of the PspAs of clades 1 to 5 were high. When the PspA2-PspA3 fusionprotein (PspA3+2) was used, the antibody titers against the PspAs of allthe clades were equivalent to or higher than those in the combined useof PspA2 and PspA3. It was also shown that immunization with a highconcentration of the PspA2-PspA3 fusion protein (PspA3+2), even in theabsence of the adjuvant, provided high antibody titers against the PspAsof all the clades. These results show that the PspA-based fusion proteinhas a potential of providing antibody titers equivalent to or higherthan those in a combined use of the two PspAs as separate proteins, doesnot require a combined use with the adjuvant to provide high antibodytiters, and thus is very useful as an antigen of pneumococcal vaccines.

Example 6: Examination on Adjuvant (1)

The effect of CpG used in combination with Alum or used alone as anadjuvant was examined.

(1) Immunization of Mice

The antigens used were the three kinds of PspA-based fusion proteinsprepared in Example 1 (PspA2+4, PspA2+5, PspA3+2), and the adjuvant usedwas a combination of CpG and Alum, or CpG alone. The following sixgroups were prepared.

-   -   0.1 μg of PspA2+4+2.5 μg of CpGK3    -   0.1 μg of PspA2+4+2.5 μg of CpGK3+5.0 μg of Alum    -   0.1 μg of PspA2+5+2.5 μg of CpGK3    -   0.1 μg of PspA2+5+2.5 μg of CpGK3+5.0 μg of Alum    -   0.1 μg of PspA3+2+2.5 μg of CpGK3    -   0.1 μg of PspA3+2+2.5 μg of CpGK3+5.0 μg of Alum

An antigen solution was prepared in LPS-free PBS and subcutaneouslyinjected into the mice for vaccination. The vaccination was performedevery week, 3 times in total. One week after the final immunization (3rdvaccination), the blood was drawn from each mouse and the serum wasseparated.

(2) ELISA

ELISA was performed in the same manner as in Example 5, and theanti-PspA antibody titers of the antisera were determined.

(3) Results

The results are shown in FIG. 8. In FIG. 8, the symbol * indicates thatthe antibody titer against the PspA-based antigen protein of a givenclade in the Alum-addition group is significantly higher at P<0.05 thanthat in the Alum-free group, and the symbol ** indicates that theAlum-addition group has a significantly higher antibody titer at P<0.01in the same comparison. In the PspA2+4 groups, under Alum-freeconditions, the antibody titers against the PspAs of clades 2, 4 and 5were high while the antibody titers against the PspAs of clades 1 and 3were low, but under Alum-addition conditions, the antibody titersagainst the PspAs of clades 1 and 3 were increased. In the PspA2+5groups, under Alum-free conditions, the antibody titers against thePspAs of clades 1 and 3 were particularly low, and even underAlum-addition conditions, the antibody titer against the clade 3 PspAwas still low. In the PspA3+2 groups, under Alum-free conditions, theantibody titer against the clade 1 PspA was low, but under Alum-additionconditions, the antibody titers against the PspAs of all the clades werehigh. These results show that, under the conditions in theseexperiments, the addition of Alum increases the specific antibody titersagainst PspAs and that the fusion type PspAs induce high specificantibody titers against the PspAs of all the clades.

Example 7: Examination on Adjuvant (2)

The effect of Alum used alone, not in combination with CpG, as anadjuvant was examined.

(1) Immunization of Mice

The antigen used was a PspA-based fusion protein prepared in Example 1(PspA3+2), and the adjuvant used was Alum. The following three groupswere prepared.

-   -   0.1 μg of PspA3+2+5.0 μg of Alum    -   0.1 μg of PspA3+2+25.0 μg of Alum    -   0.1 μg of PspA3+2+50.0 μg of Alum

An antigen solution was prepared in LPS-free PBS and subcutaneouslyinjected into the mice for vaccination. The vaccination was performedevery week, 3 times in total. One week after the final immunization (3rdvaccination), the blood was drawn from each mouse and the serum wasseparated.

(2) ELISA

ELISA was performed in the same manner as in Example 5, and theanti-PspA antibody titers of the antisera were determined.

(3) Results

The results are shown in FIG. 9. When Alum was used alone, the antibodytiters against all of the PspAs of clades 1 to 5 were high. Even whenthe dose of Alum was as low as 5.0 which was the same amount as that ina combined use with CpG, the antibody titers were almost equivalent tothose in the combined use.

Reference Example 1: PspA Family Distribution Among PneumococcalClinical Isolates in Japan

The PspA family and clade distribution was examined among 73 adultinvasive pneumococcal isolates collected in Japan. For the PspA familyand clade identification, as is the case with Example 4, an about 400-bpnucleotide sequence upstream of the proline-rich region was comparedwith the corresponding sequences in the PspA genes of which the familiesand clades were already identified.

The results are shown in Table 5. All of the 145 strains were shown tobear a family 1 or 2 PspA. Among them, 126 isolates (86.9%) included theserotypes covered by the 23-valent pneumococcal polysaccharide vaccine(PPV23) for adults, 57 isolates (39.3%) included the serotypes coveredby PCV7, and 108 isolates (74.5%) included the serotypes covered byPCV13.

TABLE 5 Serotypes and PspA clades of pneumococcal clinical isolates inJapan Coverage by PspA family and Glade of current vaccines Number. eachserotype (Number of isolates) Sero- 23- 7- 13- of Family 1 Family 2Family 3 type valent valent valent isolates Clade 1 Clade 2 Clade 3Clade 4 Clade 5 Clade 6  1 + + 1 1  3 + + 42 37 3 1 1  4 + + + 12 11 1 6A + 3 1 1 1  6B + + + 14 9 5  6C 4 1 3  7F + + 4 4  9V + + + 4 4 10A +3 3 11A + 2 1 1 12F + 2 2 14 + + + 14 12 1 1 15A 3 1 2 15B + 1 1 16 1 118B + + + 1 1 18C + + 1 1 19A + + + 7 7 19F + 5 2 2 1 20 + 1 1 22F 5 523A + + + 2 1 1 23F + 7 1 6 33 1 1 34 1 1 35 3 3 38 1 1 Total 145 78 642 10 9 0 (53.8%) (4.1%) (29%) (6.9%) (6.2%)

The present invention is not limited to the particular embodiments andexamples described above, and various modifications can be made withinthe scope of the appended claims. Other embodiments provided by suitablycombining technical means disclosed in separate embodiments of thepresent invention are also within the technical scope of the presentinvention. All the academic publications and patent literature cited inthe description are incorporated herein by reference.

The invention claimed is:
 1. A pneumococcal vaccine for parenteraladministration comprising a fusion protein comprising a full-lengthfamily 1, clade 2 pneumococcal surface protein A (PspA) or a fragmentthereof, and a full-length family 2 PspA or a fragment thereof, whereinthe full-length family 1, clade 2 PspA or a fragment thereof and thefull-length family 2 PspA or a fragment thereof each comprises the wholeof a proline-rich region and the whole or part of an α-helical regionadjacent thereto, the fragment consists of 108 amino acid residues ormore, and the fusion protein possesses the ability to induce protectiveimmunity against pneumococcal infections in a living body, the fusionprotein being any one of the following (1) to (3): (1) a fusion proteinat least comprising a full-length family 1, clade 2 PspA or a fragmentthereof, and a full-length family 2, clade 3 PspA or a fragment thereof,(2) a fusion protein at least comprising a full-length family 1, clade 2PspA or a fragment thereof, and a full-length family 2, clade 4 PspA ora fragment thereof, and (3) a fusion protein at least comprising afull-length family 1, clade 2 PspA or a fragment thereof, and afull-length family 2, clade 5 PspA or a fragment thereof.
 2. Thepneumococcal vaccine according to claim 1, wherein the fusion protein isany one of the following (4) to (6): (4) a fusion protein consisting ofa fragment of family 1, clade 2 PspA, and fragment of family 2, clade 3PspA, wherein the fragment consists of the whole of a proline-richregion and the whole or part of an α-helical region adjacent thereto,(5) a fusion protein consisting of a fragment of family 1, clade 2 PspA,and a fragment of family 2, clade 4 PspA, wherein the fragment consistsof the whole of a proline-rich region and the whole or part of anα-helical region adjacent thereto, and (6) a fusion protein consistingof a fragment of family 1, clade 2 PspA, and a fragment of family 2,clade 5 PspA, wherein the fragment consists of the whole of aproline-rich region and the whole or part of an α-helical regionadjacent thereto.
 3. The pneumococcal vaccine according to claim 1,wherein the family 1, clade 2 PspA is from a pneumococcal strainselected from the group consisting of D39, WU2, E134, EF10197, EF6796,BG9163 and DBL5.
 4. The pneumococcal vaccine according to claim 1,wherein the family 2, clade 3 PspA is from a pneumococcal strain TIGR4,BG8090 or AC122, the family 2, clade 4 PspA is from a pneumococcalstrain EF5668, BG7561, BG7817 or BG11703, and the family 2, clade 5 PspAis from a pneumococcal strain ATCC6303 or KK910.
 5. The pneumococcalvaccine according to claim 1, wherein the fusion protein consists of anamino acid sequence at least 90% identical to SEQ ID NO: 1, 3 or
 5. 6. Apneumococcal vaccine for parenteral administration comprising any one ofthe following (i) to (iii): (i) a combination of only a fragment offamily 1, clade 2 PspA and a fragment of family 2, clade 3 PspA, whereinthe fragment consists of the whole of a proline-rich region and thewhole or part of an α-helical region adjacent thereto, (ii) acombination of only a fragment of family 1, clade 2 PspA and a fragmentof family 2, clade 4 PspA, wherein the fragment consists of the whole ofa proline-rich region and the whole or part of an α-helical regionadjacent thereto, and (iii) a combination of only a fragment of family1, clade 2 PspA and a fragment of family 2, clade 5 PspA, wherein thefragment consists of the whole of a proline-rich region and the whole orpart of an α-helical region adjacent thereto.
 7. The pneumococcalvaccine according to claim 1, wherein the fusion protein is any one ofthe following (4) to (6): (4) a fusion protein composed of a fragmentconsisting of the whole of α-helical and proline-rich regions of a D39PspA, and a fragment consisting of the whole of α-helical andproline-rich regions of an EF5668 PspA, (5) a fusion protein composed ofa fragment consisting of the whole of α-helical and proline-rich regionsof a D39 PspA, and a fragment consisting of the whole of α-helical andproline-rich regions of an ATCC6303 PspA, and (6) a fusion proteincomposed of a fragment consisting of the whole of α-helical andproline-rich regions of a WU2 PspA, and a fragment consisting of thewhole of α-helical and proline-rich regions of a TIGR4 PspA.
 8. Thepneumococcal vaccine according to claim 7, wherein the fusion protein isthat described in the above (6).
 9. The pneumococcal vaccine accordingto claim 5, wherein the fusion protein consists of an amino acidsequence at least 90% identical to SEQ ID NO:
 5. 10. A method forprevention or treatment of pneumococcal disease, comprising parenterallyadministering, to an animal, an effective amount of a fusion proteincomprising a full-length family 1, clade 2 PspA or a fragment thereof,and a full-length family 2 PspA or a fragment thereof, wherein thefull-length family 1, clade 2 PspA or a fragment thereof and thefull-length family 2 PspA or a fragment thereof each comprises the wholeof a proline-rich region and the whole or part of an α-helical regionadjacent thereto, the fragment consists of 108 amino acid residues ormore, and the fusion protein possesses the ability to induce protectiveimmunity against pneumococcal infections in a living body, the fusionprotein being any one of the following (1) to (3): (1) a fusion proteinat least comprising a full-length family 1, clade 2 PspA or a fragmentthereof, and a full-length family 2, clade 3 PspA or a fragment thereof,(2) a fusion protein at least comprising a full-length family 1, clade 2PspA or a fragment thereof, and a full-length family 2, clade 4 PspA ora fragment thereof, and (3) a fusion protein at least comprising afull-length family 1, clade 2 PspA or a fragment thereof, and afull-length family 2, clade 5 PspA or a fragment thereof.
 11. A methodfor prevention or treatment of pneumococcal disease, comprisingparenterally administering, to an animal, effective amounts of at leastthe following components: a full-length family 1, clade 2 PspA or afragment thereof, and a full-length family 2 PspA selected from thegroup consisting of clade 3, 4 and 5 PspAs, or a fragment thereof,wherein the full-length family 1, clade 2 PspA or a fragment thereof,and the full-length family 2 PspA selected from the group consisting ofclade 3, 4 and 5 PspAs, or a fragment thereof each comprises the wholeof a proline-rich region and the whole or part of an α-helical regionadjacent thereto, the fragment consists of 108 amino acid residues ormore, and the components possess the ability to induce protectiveimmunity against pneumococcal infections in a living body.
 12. Thepneumococcal vaccine according to claim 1, further comprising anadjuvant.
 13. The pneumococcal vaccine according to claim 1, furthercomprising a vaccine component against a pathogen other thanpneumococci.
 14. The method according to claim 10, wherein the fusionprotein is any one of the following (4) to (6): (4) a fusion proteinconsisting of a fragment of family 1, clade 2 PspA, and a fragment offamily 2, clade 3 PspA, wherein the fragment consists of the whole of aproline-rich region and the whole or part of an α-helical regionadjacent thereto, (5) a fusion protein consisting of a fragment offamily 1, clade 2 PspA, and h a fragment of family 2, clade 4 PspA,wherein the fragment consists of the whole of a proline-rich region andthe whole or part of an α-helical region adjacent thereto, and (6) afusion protein consisting of a fragment of family 1, clade 2 PspA, and afragment of family 2, clade 5 PspA, wherein the fragment consists of thewhole of a proline-rich region and the whole or part of an α-helicalregion adjacent thereto.
 15. The method according to claim 10, whereinthe fusion protein consists of an amino acid sequence at least 90%identical to SEQ ID NO: 1, 3 or
 5. 16. The method according to claim 10,wherein the fusion protein is any one of the following (4) to (6): (4) afusion protein composed of a fragment consisting of the whole ofα-helical and proline-rich regions of a D39 PspA, and a fragmentconsisting of the whole of α-helical and proline-rich regions of anEF5668 PspA, (5) a fusion protein composed of a fragment consisting ofthe whole of α-helical and proline-rich regions of a D39 PspA, and afragment consisting of the whole of α-helical and proline-rich regionsof an ATCC6303 PspA, and (6) a fusion protein composed of a fragmentconsisting of the whole of α-helical and proline-rich regions of a WU2PspA, and a fragment consisting of the whole of α-helical andproline-rich regions of a TIGR4 PspA.
 17. The method according to claim11, wherein the fragment of family 1, clade 2 PspA consists of an aminoacid sequence at least 90% identical to SEQ ID NO: 25 or 26, thefragment of family 2, clade 3 PspA consists of an amino acid sequence atleast 90% identical to SEQ ID NO: 27, the fragment of family 2, clade 4PspA consists of an amino acid sequence at least 90% identical to SEQ IDNO: 28, and the fragment of family 2, clade 5 PspA consists of an aminoacid sequence at least 90% identical to SEQ ID NO: 29.